Application of short peptide entrapped by lung-targeted liposome and used for blocking combination of PKM2-FOXO3A in preparation of medicine for treating stroke-related lung injury

Abstract

The invention belongs to the technical field of biological medicine, and particularly relates to application of a short peptide entrapped by lung-targeted liposome and used for blocking combination of PKM2-FOXO3A in preparation of a medicine for treating stroke-related lung injury, the amino acid sequence of the short peptide is shown as SEQ ID NO.1, the SEQ ID NO.1 RKGEDREGKR and Pe (at) P-Lipo can be used for remarkably relieving MCAO mouse lung tissue pathological injury, and can be used for preparing a medicine for treating stroke-related lung injury. According to the present invention, with the application of the cerebral ischemia injury in the lung, the total reactive oxygen species (ROS) level in the lung tissue and the mitochondrial ROS level, the reduction of the ATP content can be reversed, the level of the lung tissue inflammation-related protein can be reduced, and the enrichment of PKM2, FOXO3A and TXNIP in the mitochondria can be reduced so as to alleviate the lung tissue oxidative stress and the mitochondrial injury caused by the cerebral ischemia injury, and provide the lung protection effect;

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biological medicine, and particularly relates to application of a lung-targeting liposome loaded with a short peptide for blocking combination of PKM2-FOXO3A in preparation of a drug for stroke-related lung injury. BACKGROUND

[0002] The statements herein merely provide background technology related to the present application, and do not necessarily constitute prior art.

[0003] Ischemic stroke is an acute cerebrovascular disease with high mortality and high disability rate. Although reperfusion therapy such as thrombolysis and thrombectomy can effectively restore cerebral blood flow and reduce mortality in the acute stage of stroke, many survivors still suffer from severe systemic complications, among which acute lung injury is particularly common and critical, significantly affecting the prognosis of patients and increasing the risk of death. At present, the specific pathogenesis of lung injury after stroke has not been fully elucidated, and there is a lack of specific prevention and treatment methods for this complication in clinical practice, which has become a major challenge and unmet clinical need in the comprehensive treatment of stroke. SUMMARY

[0004] In view of the deficiencies in the prior art, the purpose of the present application is to provide application of a lung-targeting liposome loaded with a short peptide for blocking combination of PKM2-FOXO3A in preparation of a drug for stroke-related lung injury.

[0005] In order to achieve the above-mentioned purpose, the present application is realized by the following technical scheme: In a first aspect, the present application provides application of a lung-targeting liposome loaded with a short peptide for blocking combination of PKM2-FOXO3A in preparation of a drug for stroke-related lung injury, wherein the amino acid sequence of the short peptide is shown as SEQ ID NO. 1, and SEQ ID NO. 1 is RKGEDREGKR.

[0006] In a second aspect, the present application provides a preparation method of a lung-targeting liposome loaded with a short peptide for blocking combination of PKM2-FOXO3A, comprising the following steps: Dissolve dipalmitoyl phosphatidylcholine (DPPC), DSPE-PEG2000-FITC and cholesterol in an organic solvent in a certain proportion, then evaporate the organic solvent to obtain a thin film layer; Disperse the thin film layer in a phosphate buffer to obtain a liposome with lung targeting ability; Ice-bath homogenate emulsify the short peptide and the liposome to obtain a lung-targeting liposome loaded with the interfering peptide.

[0007] DSPE-PEG2000-FITC (1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-polyethylene glycol 2000-fluorescein) is a functionalized phospholipid derivative, which combines the biocompatibility of phospholipids, the stability of polyethylene glycol (PEG) and the fluorescence labeling ability of fluorescein (FITC).

[0008] In a third aspect, the present application provides a lung-targeted liposome of a short peptide blocking the combination of PKM2 and FOXO3A, which is prepared by the preparation method.

[0009] The beneficial effects achieved by one or more embodiments of the present application are as follows: The Pe@P-Lipo treatment can significantly alleviate the pathological damage of lung tissue in MCAO mice, reverse the decrease of ATP content, reduce the levels of inflammation-related proteins in lung tissue, and reduce the enrichment of PKM2, FOXO3A and TXNIP in mitochondria, thereby relieving oxidative stress and mitochondrial damage in lung tissue caused by cerebral ischemic injury and playing a lung protection role. This strategy specifically targets the newly discovered harmful signal pathway of the "brain-lung axis", and solves the problems of systemic complications that cannot be alleviated by current reperfusion therapy and toxic side effects that may be caused by systemic administration through precise delivery and intervention. BRIEF DESCRIPTION OF DRAWINGS

[0010] The drawings accompanying the specification of the present application form a part thereof, serve to provide further understanding of the present application, and together with the description, explain the present application. The present application is shown by way of illustration in the drawings and by the description given below, and is not intended to be limited thereby.

[0011] Figure 1 In the figure, (A) is a design diagram of the interference peptide sequence; the right side is an enlarged view of the left side block; (B) is a diagram in which yellow represents amino acid residues on PKM2 covered by sequence 3, including R120 and K206, and green circles represent R120 and K206 on PKM2; (C) is a diagram of the electrostatic potential energy of PKM2; blue represents positive charge, and red represents negative charge; (D) is a diagram of CoIP verification of the inhibition efficiency of interference peptide #3 on the interaction between PKM2 and FOXO3A; (E) is a diagram of the effect of interference peptide #3 on the levels of PKM2, FOXO3A and TXNIP in mitochondria of alveolar epithelial cells MLE-12; Figure 2In the specific embodiments, (A) is a transmission electron microscope image of Pe@P-Lipo and P-Lipo; (B) is a particle size analysis chart of Pe@P-Lipo and P-Lipo; (C) is a Zeta potential chart of Pe@P-Lipo and P-Lipo; (D) is a fluorescence chart of Pe@P-Lipo and P-Lipo; (E) verifies the targeting of P-Lipo to lung tissue; Figure 3 In the specific embodiments, (A) is a flow chart of the experimental design; (B) is a lung tissue H&E staining chart of MCAO mice; (C) is a lung tissue ATP level detection chart; (D) is a DHE staining chart of lung tissue of MCAO mice; (E) is a MitoSOX staining chart of lung tissue of MCAO mice; (F) is a lung tissue inflammatory factor protein level detection chart of MCAO mice; (G) is a lung tissue mitochondrial protein detection chart of MCAO mice. Figure 4 In the specific embodiments, (A) is a comparison chart of hemolysis test results; (B) is a columnar comparison chart of hemolysis test results; (C) is a columnar chart of serum AST biochemical index detection of MCAO mice; (D) is a columnar chart of serum ALT biochemical index detection of MCAO mice; (E) is a columnar chart of serum BUN biochemical index detection of MCAO mice; (F) is a H&E staining chart of important organs (heart, liver, spleen, kidney) of MCAO mice. DETAILED DESCRIPTION

[0012] It should be noted that the following detailed description is exemplary and is intended to provide further explanation of the application. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.

[0013] The inventors have found that extracellular vesicles derived from activated neurons on the lesion side of stroke can act as a "messenger" to transmit the injury signal of the brain to peripheral organs. The inventors have found for the first time that M2-type pyruvate kinase (PKM2) of neuronal origin is a key molecule that mediates this harmful "brain-lung axis" communication. Specifically, PKM2 enters the circulatory system through extracellular vesicles, acts on alveolar epithelial cells, and induces phosphorylation of the S252 site of transcription factor FOXO3A by binding to FOXO3A, thereby promoting the translocation of the FOXO3A / TXNIP complex to mitochondria, ultimately leading to mitochondrial dysfunction of alveolar epithelial cells and triggering acute lung injury. The revelation of this new mechanism provides a completely new target for intervention of stroke complications.

[0014] However, how to achieve precise and efficient intervention on the target is a big problem. Inhibition of PKM2 by systemic administration may interfere with the normal metabolic function of other tissues, causing unpredictable side effects. Therefore, the development of a technology that can specifically deliver therapeutic drugs to the lungs and precisely block the PKM2 / FOXO3A interaction is of great significance for effectively treating lung injury after stroke while minimizing systemic toxic side effects.

[0015] Based on this, the application provides a lung-targeting liposome loaded with a short peptide blocking the combination of PKM2-FOXO3A in the preparation of a drug for stroke-related lung injury, wherein the amino acid sequence of the short peptide is shown as SEQ ID NO. 1, and SEQ ID NO. 1 is RKGEDREGKR.

[0016] The targeting of the liposome enables efficient enrichment of the drug at the lung lesion site, avoids affecting other tissues in the body, and improves treatment specificity and safety; secondly, the newly discovered pathogenic signaling pathway is precisely cut off by the interference peptide, which prevents mitochondrial damage of alveolar epithelial cells from the root, thereby achieving effective prevention and treatment of lung injury induced by stroke. The application not only provides a new candidate drug for solving the major complication of stroke, but also provides an innovative paradigm of "targeted delivery + precise intervention" for intervening in the long-range communication mechanism between organs, which has important clinical translation value and application prospect.

[0017] In the second aspect, the application provides a preparation method of a lung-targeting liposome loaded with a short peptide blocking the combination of PKM2-FOXO3A, comprising the following steps: Dissolve the lipids DPPC, DSPE-PEG2000-FITC and cholesterol in an organic solvent in a certain proportion, then evaporate the organic solvent to obtain a thin film layer; Disperse the thin film layer in a phosphate buffer to obtain a liposome with lung targeting ability; Ice-bath homogenate emulsify the short peptide and the liposome to obtain a lung-targeting liposome loaded with the short peptide.

[0018] DPPC (dipalmitoyl phosphatidylcholine) is the main lipid component of the bilayer membrane of the liposome, which provides the basic skeletal structure of the liposome and is one of the core materials for forming a liposome with lung targeting ability; As a functionalized phospholipid derivative, DSPE-PEG2000-FITC has the following functions: ① the phospholipid part helps to embed the liposome membrane; ② the PEG (polyethylene glycol) part improves the stability of the liposome and reduces clearance; and ③ the FITC (fluorescein) part realizes fluorescence labeling and is used to verify the targeting of the liposome to the lung tissue; Cholesterol regulates the fluidity of liposome membranes, enhances membrane stability, and prevents liposome fusion or rupture, making it a key auxiliary component for maintaining the structural stability of liposomes.

[0019] Ice bath homogenization and emulsification are performed to maintain a low-temperature environment. On the one hand, this protects the membrane structure stability of liposomes, preventing liposome fusion or rupture caused by high temperatures. On the other hand, it prevents interfering peptides (short peptide biomolecules) from denaturing due to temperature increases, ensuring that their biological activity is not destroyed. Mechanical homogenization ensures that the interfering peptides are fully mixed with the liposomes, promoting uniform loading of the interfering peptides into the interior or surface of the liposomes, improving drug loading efficiency, and forming lung-targeting liposomes with uniform particle size and stable dispersion, ensuring the consistency and effectiveness of the formulation.

[0020] In some embodiments, the mass ratio of DPPC, DSPE-PEG2000-FITC and cholesterol is 6-10:0.8-1.2:0.8-1.2.

[0021] Preferably, the mass ratio of DPPC, DSPE-PEG2000-FITC and cholesterol is 7-9:0.9-1.1:0.9-1.1.

[0022] Further preferred, the mass ratio of DPPC, DSPE-PEG2000-FITC and cholesterol is 8:1:1.

[0023] In some embodiments, the organic solvent is chloroform, methanol, ethanol, dichloromethane, tert-butanol, or a chloroform-methanol mixture.

[0024] In some embodiments, the method for removing organic solvents by evaporation is vacuum evaporation at a temperature of 25-35°C.

[0025] The core components of liposomes (such as DPPC) form a bilayer membrane framework. Low temperature can avoid the disordered arrangement of lipid molecules, damage to membrane structure, or premature fusion and rupture of liposomes caused by high temperature, ensuring the formation of a complete basic structure of lung-targeting liposomes. Low temperature can reduce the risk of oxidation or denaturation of lipids such as DPPC and cholesterol during the evaporation process, maintaining their biocompatibility and functional integrity. The vacuum environment lowers the boiling point of organic solvents (chloroform), and the solvent can still be effectively volatilized and removed at low temperature, while avoiding the adverse effects of high temperature on the lipid system.

[0026] In some embodiments, the pH of the phosphate buffer solution is 7-7.5.

[0027] Thirdly, the present invention provides a lung-targeting liposome that blocks the binding of PKM2-FOXO3A to a short peptide, prepared by the aforementioned preparation method.

[0028] The present invention will be further described below with reference to the embodiments.

[0029] Example 1 Experimental procedure: 1. MCAO Model: The experimental animals were male C57BL / 6J mice, weighing 22-25g. Animals were purchased from the Experimental Animal Center of Shandong University. Animal experiments were conducted in accordance with the International Guidelines for Animal Research provided by the Council for Medical Sciences, and the procedures were approved by the Animal Ethics and Welfare Committee of Shandong University. All experimental personnel were trained according to the guidelines and rules of the Committee on Animal Care and Use.

[0030] A middle cerebral artery (MCAO) model was established using the suture embolization method. In short, mice were anesthetized with 2% isoflurane. The right carotid artery, internal carotid artery, and external carotid artery were isolated. A suture embolization device (Jialing, Guangzhou, China) was then inserted into the internal carotid artery until resistance was felt, occluding the middle cerebral artery for 2 hours before removal to achieve reperfusion. During the procedure, an adjustable heating pad was used to maintain a body temperature of 37°C. In the sham-operated group (Sham group), the right internal carotid artery was exposed after anesthesia but not embolized.

[0031] 2. Preparation of Pe@P-Lipo: (1) Lipids DPPC, DSPE-PEG2000-FITC and cholesterol were dissolved in chloroform (5 mL) at a mass ratio of 8:1:1. The solvent was evaporated under vacuum at 30 °C and 100 rpm / min (2 h) to remove the chloroform. The uniform film layer was then dispersed in 2 mL of sterile phosphate-buffered saline (PBS; pH 7.4, 0.01 M) solution to form liposomes (P-Lipo) with lung-targeting capabilities.

[0032] (2) The mixture of interfering peptide and P-Lipo was emulsified on ice using an ultrasonic homogenizer for 150 seconds to obtain lung-targeting liposomes loaded with interfering peptide (Pe@P-Lipo). The nanosuspension was stored at 4°C for subsequent experiments.

[0033] 20 mg of soybean lecithin and 5 mg of cholesterol were dispersed in 10 mL of ethanol to synthesize conventional liposomes (C-Lipo) using the method described above, and then redissolved in sterile PBS. C-Lipo was labeled with fluorescein isothiocyanate (FITC).

[0034] 3. Experimental grouping: Mice were randomly divided into two groups: the MCAO+Pe@P-Lipo group and the MCAO+P-Lipo group. 24 h after MCAO injury, lung tissue pathology, ROS levels, inflammation-related protein levels, and mitochondrial PKM2, FOXO3A, and TXNIP protein levels were measured.

[0035] 4. Western blotting analysis: Lung tissue was homogenized in RIPA buffer containing protease / phosphatase inhibitors and PMSF for 15 min, then blotting at 12,000 rpm for 10 min (4°C). Protein concentration was determined using a BCA protein assay kit. Chemiluminescent signals were generated using an ECL kit and then detected using a Tanon imaging system.

[0036] 5. H&E staining: Mice were sacrificed 24 h after MCAO injury, and paraffin sections of mouse lung tissue were obtained for H&E staining to observe the pathological damage of the lung tissue.

[0037] 6. Detection of reactive oxygen species (ROS) levels: Mice were sacrificed 24 h after MCAO injury, and frozen sections of mouse lung tissue were obtained. The tissue was incubated with a fluorescent probe (DHE or MitoSOX) for 30 min (37 ℃). Images were acquired using a fluorescence microscope and quantitative fluorescence analysis was performed.

[0038] 7. Detection of mitochondrial ATP content: Mice were sacrificed 24 h after MCAO injury, and fresh lung tissue was taken from the mice. The ATP content of the lung tissue of MCAO mice was detected using an ATP detection kit.

[0039] 8. Mitochondrial protein detection: Mice were sacrificed 24 h after MCAO injury. Mitochondria were extracted from mouse lung tissue using a mitochondrial extraction kit, and then mitochondrial proteins were obtained by lysis using mitochondrial lysis buffer. Finally, the content of mouse mitochondrial proteins (PKM2, FOXO3A, and TXNIP) was detected using Western blotting.

[0040] Experimental results: 1. In vitro verification of the efficiency of interfering peptides in inhibiting the interaction between PKM2 and FOXO3A.

[0041] Figure 1In the diagram, A represents the design of the interfering peptide sequences (Sequence 1: RKGEDDEGKR (SEQ ID NO. 2); Sequence 2: RKGGEDDEGGKR (SEQ ID NO. 3); Sequence 3 (#3): RKGEDREGKR (SEQ ID NO. 1); the right side is an enlarged version of the left box; the docking results of sequence 3 and PKM2 show that sequence 3 can bind to the pocket of PKM2, which is the key interaction region between PKM2 and FOXO3A. B, yellow represents the amino acid residues on PKM2 covered by sequence 3, including R120 and K206; the green circles represent R120 and K206 on PKM2; C is the electrostatic potential energy diagram of PKM2; blue represents positive values ​​(positive charge), and red represents negative values ​​(negative charge), defined as the electrostatic potential energy of a positive charge from infinity to any point on the graph. D is the CoIP verification of the interfering peptide #. 3. Inhibition efficiency of interfering peptide #3 on PKM2-FOXO3A interaction. E represents the effect of interfering peptide #3 on the levels of PKM2, FOXO3A, and TXNIP in the mitochondria of MLE-12 alveolar epithelial cells. These data indicate that interfering peptide sequence 3 can effectively inhibit PKM2-FOXO3A interaction.

[0042] 2. Characterization and lung targeting of Pe@P-Lipo Figure 2 In the image, A shows transmission electron microscopy (TEM) images of Pe@P-Lipo and P-Lipo. B shows particle size analysis of Pe@P-Lipo and P-Lipo. C shows the zeta potentials of Pe@P-Lipo and P-Lipo. D shows the fluorescence images of Pe@P-Lipo and P-Lipo. E confirms the targeting of P-Lipo to lung tissue.

[0043] 3. Pe@P-Lipo can alleviate lung tissue damage in MCAO mice. Figure 3In the diagram, A is the experimental design flowchart. MCAO mice were nebulized with Pe@P-Lipo and P-Lipo 30 min after surgery, and lung injury was assessed 24 h after reperfusion. B shows H&E staining of MCAO mouse lung tissue, indicating that Pe@P-Lipo treatment significantly reduced pathological damage in the lung tissue of MCAO mice compared to the P-Lipo treatment group. C shows the detection of ATP levels in lung tissue, indicating that Pe@P-Lipo treatment significantly increased ATP production in the lung tissue of MCAO mice compared to the P-Lipo treatment group. D shows DHE staining of MCAO mouse lung tissue, indicating that Pe@P-Lipo treatment significantly reduced superoxide anion levels in the lung tissue of MCAO mice compared to the P-Lipo treatment group. E shows MitoSOX staining of MCAO mouse lung tissue, indicating that Pe@P-Lipo treatment significantly reduced mitochondrial ROS levels in the lung tissue of MCAO mice compared to the P-Lipo treatment group. F shows the detection of inflammatory cytokine protein levels in the lung tissue of MCAO mice. The results indicate that, compared with the P-Lipo treatment group, Pe@P-Lipo treatment significantly reduced the inflammatory response in the lung tissue of MCAO mice, as evidenced by decreased levels of inflammatory cytokines TNF-α, iNOS, and IL-1β. G shows the detection of mitochondrial proteins in the lung tissue of MCAO mice. The results show that, compared with the P-Lipo treatment group, Pe@P-Lipo treatment significantly reduced the accumulation of PKM2, FOXO3A, and TXNIP in the mitochondria of MCAO mouse lung tissue. Statistical value = Mean ± Standard deviation; p A value <0.05 was considered statistically significant. Data were analyzed using Student's t-test.

[0044] 4. Pe@P-Lipo exhibits good biocompatibility. Figure 4 In the figures, A and B represent the results of the hemolysis test, showing that the dose of Pe@P-Lipo used in this invention did not cause significant hemolytic reaction, indicating that Pe@P-Lipo has good blood compatibility. CE represents the detection of serum biochemical indicators in MCAO mice, showing that Pe@P-Lipo treatment had no significant effect on serum AST, ALT, and BUN levels in MCAO mice, indicating that Pe@P-Lipo has no significant hepatotoxicity or nephrotoxicity in MCAO mice. F represents the H&E staining of vital organs (heart, liver, spleen, and kidney) in MCAO mice, showing that Pe@P-Lipo treatment did not cause significant structural damage to vital organs in MCAO mice. These data demonstrate that Pe@P-Lipo has good biocompatibility. Statistical value = mean ± standard deviation.

[0045] The above experiments fully demonstrate that the lung-targeting liposomes provided by this invention can efficiently deliver therapeutic interfering peptides to the lungs, and effectively reverse downstream pathogenic signals and improve mitochondrial function by specifically blocking the interaction between PKM2 and FOXO3A. Ultimately, it significantly reduces acute lung injury induced by ischemic stroke in animal models, showing good application prospects.

[0046] Example 2 Preparation of Pe@P-Lipo: (1) Lipids DPPC, DSPE-PEG2000-FITC, and cholesterol were dissolved in chloroform (5 mL) at a mass ratio of 7:1:1. The solvent was evaporated under vacuum at 25 °C and 100 rpm / min (2 h) to remove the chloroform. The uniform film layer was then dispersed in 2 mL of sterile phosphate-buffered saline (PBS; pH 7.4, 0.01 M) solution to form liposomes (P-Lipo) with lung-targeting capabilities.

[0047] (2) The mixture of interfering peptide and P-Lipo was emulsified on ice using an ultrasonic homogenizer for 130 seconds to obtain lung-targeting liposomes loaded with interfering peptide (Pe@P-Lipo). The nanosuspension was stored at 4°C for subsequent experiments.

[0048] Example 3 Preparation of Pe@P-Lipo: (1) Lipids DPPC, DSPE-PEG2000-FITC, and cholesterol were dissolved in chloroform (5 mL) at a mass ratio of 9:1.2:1. The solvent was evaporated under vacuum at 35 °C and 100 rpm / min (2 h) to remove the chloroform. The uniform film layer was then dispersed in 2 mL of sterile phosphate-buffered saline (PBS; pH 7.4, 0.01 M) solution to form liposomes (P-Lipo) with lung-targeting capabilities.

[0049] (2) The mixture of interfering peptide and P-Lipo was emulsified on ice using an ultrasonic homogenizer for 160 seconds to obtain lung-targeting liposomes loaded with interfering peptide (Pe@P-Lipo). The nanosuspension was stored at 4°C for subsequent experiments.

[0050] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. The application of a short peptide encapsulated in a lung-targeting liposome to block PKM2-FOXO3A binding in the preparation of drugs for stroke-related lung injury, characterized in that: The amino acid sequence of the short peptide is shown in SEQ ID NO. 1, SEQ ID NO. 1: RKGEDREGKR.

2. A method for preparing lung-targeting liposomes loaded with a short peptide that blocks PKM2-FOXO3A binding, characterized in that: Includes the following steps: Lipid DPPC, DSPE-PEG2000-FITC and cholesterol were dissolved in an organic solvent in a certain proportion, and then the organic solvent was evaporated to remove the organic solvent to obtain a thin film layer. The thin film layer was dispersed in phosphate buffer to obtain liposomes with lung-targeting ability; The short peptide of claim 1 and the liposome were homogenized and emulsified in an ice bath to obtain lung-targeting liposomes loaded with the short peptide.

3. The method for preparing lung-targeting liposomes loaded with a short peptide that blocks PKM2-FOXO3A binding according to claim 2, characterized in that: The mass ratio of DPPC, DSPE-PEG2000-FITC and cholesterol is 6-10:0.8-1.2:0.8-1.

2.

4. The method for preparing lung-targeting liposomes loaded with a short peptide that blocks PKM2-FOXO3A binding according to claim 3, characterized in that: The mass ratio of DPPC, DSPE-PEG2000-FITC and cholesterol was 7-9:0.9-1.1:0.9-1.

1.

5. The method for preparing lung-targeting liposomes loaded with a short peptide that blocks PKM2-FOXO3A binding according to claim 4, characterized in that: The mass ratio of DPPC, DSPE-PEG2000-FITC and cholesterol is 8:1:

1.

6. The method for preparing lung-targeting liposomes loaded with a short peptide that blocks PKM2-FOXO3A binding according to claim 2, characterized in that: The organic solvent is chloroform, methanol, ethanol, dichloromethane, tert-butanol, or a chloroform-methanol mixture.

7. The method for preparing lung-targeting liposomes loaded with a short peptide that blocks PKM2-FOXO3A binding according to claim 2, characterized in that: The method for removing organic solvents by evaporation is vacuum evaporation at a temperature of 25-35℃.

8. The method for preparing lung-targeting liposomes loaded with a short peptide that blocks PKM2-FOXO3A binding according to claim 7, characterized in that: Stirring is performed continuously during the evaporation process to remove the organic solvent.

9. The method for preparing lung-targeting liposomes loaded with a short peptide that blocks PKM2-FOXO3A binding according to claim 2, characterized in that: The pH value of the phosphate buffer solution is 7-7.

5.

10. A lung-targeting liposome containing a short peptide that blocks PKM2-FOXO3A binding, characterized in that: It is prepared by any one of the preparation methods described in claims 2-9.

Images